The Hungarian patent specification No. 185,263 discloses the peptides Arg Lys Asp and Arg Lys Asp Val and their action influencing the immune system. Several publications describe their wide-range in vitro and in vivo immunological and immunopharmacological effects: see e.g. Hoope Seyler's Z. Physiol. Chem., 364, 933. (1983); J. Immunopharmacol., 7, 67 (1985); Int. J. lmmunopharmacol., 8, 167 (1986); Immunpharmacol. Immunotoxicol., 9, 1 (1987).
With the knowledge of the advantageous biological results detailed toxicological and clinical examination of these two peptides has been commenced. For the preparation of a clinically usable drug the synthesis of the peptides has to be carried out on industrial scale of the magnitude of several kilogramms so that high purity products be prepared which correspond to the strict stipulations determined for pharmaceutical compositions.
During the synthesis of the two peptides described in the Hungarian patent specification No. 185,263 the amino and carboxyl groups are protected by benzyl type protecting groups and the quanidino group of arginine is protected by a nitro group. In the last step all the protecting groups are removed by catalytic hydrogenation. The conditions under which this combination of the protecting groups works encumber the extension of the reaction and/or its realization on an obtained by hydrogenation is far below the present requirements. Accordingly, a new synthesis route was elaborated.
In the Hungarian patent application No. 3037/88 published under No. T/50195 only a tert-butyl-type protecting group is used during the synthesis of the structural and optical isomers of the peptides of the present invention and the guanidino group of arginine is protected by protonation. The protecting groups are removed in one step by using trifluoroacetic acid in a known manner. The synthesis route described in the patent application No. 3037/88 eliminates a large part of the side reactions of the synthesis described in the Hungarian patent specification No. 183,263 and the product obtained is more pure. However, the extension and the industrial scale realization of the process is expensive, involves several technical and environmental problems and spoils the quality.
In human therapy a peptide purity of more than 97% is generally acceptable and the quantity of a single contamination may surpass 0.5% only if its structure is known and its innocuity has been proved by toxicological tests.
According to the processes described in the Hungarian patent specification No. 185,263 and patent application No. 3037/88 the tripeptide Arg Lys Asp and the tetrapeptide Arg Lys Asp Val were separated and purified either after ion exchange by lyophilization or after evaporation by ethanolic treatment.
In both processes the tripeptide Arg Lys Asp and the tetrapeptide Arg Lys Asp Val were isolated in amorphous state. Both amorphous and air dried substances contain 70 to 85% of a peptide, the rest consists of water, acetic acid and ethanol. Lyophilized substances generally contain 15% of acetic acid. Both the water and the acetic acid content significantly depends on the technological circumstances (starting substance concentration, lyophilization program, drying time and temperature). The solvent content of the substance isolated by ethanol after the ion exchange reflects actively to the changes in the technological parameters and it did not succeed in removing the ethanol from some amorphous samples even by modifying the drying parameters within wide ranges. Their amorphous structure can also be proved by x-ray diffraction picture made with a powdery form. No sharp peaks showing crystalline structure can be observed. The thermogravimetric measurements and differential calorimetric test unambiguously prove that the peptides in question bind a part of the accompanying solvent by adsorption and thus their quantity and ratio varies in very wide ranges, presumably due to smaller undetermined changes of the technological parameters and e.g. by the changing moisture content of the surroundings. The last effect could have unambigously been proved by a thermogravimetric test. The slightly dissected wide stripe between 1700 and 1400 cm.sup.-1 in the infrared spectrum shows the presence of water bound only by adsorption. Because of the amorphous state of the compounds even the quantity of the accompanying acetic acid is not stoichiometric and the quantity of the water and ethanol also fluctuates within wide ranges. In some countries the official stipulations for pharmaceutical starting substances allow neither a fluctuation of the quantity of the accompanying solvents within wide ranges nor the quantity of ethanol reaching 6 to 7%. The fluctuating peptide content of the products makes the adjustment of the desired dose uncertain during the preparation of the pharmaceutical compositions.
No crystalline substances of stoichiometric composition could be prepared by the known processes either through reprecipitation or treatment with a solvent of the oil obtained after ion exchange or of the solid, amorphous substance obtained from said oil.